Project Summary/Abstract Pathogenic Rickettsia species include Gram-negative bacteria known to cause human infections, namely spotted fever (R. rickettsii and R. conorii) and typhus (R. prowazekii and R. typhi), with serious morbidity and mortality. Tropism for microvascular endothelium, rapid escape from the phagosome, and growth/replication in the nutrient-rich cytosol as obligate intracellular parasites leading to vascular inflammation and dysfunction/damage constitute the salient features of rickettsial pathogenesis. Rickettsioses are vector-borne infections transmitted in nature by infected arthropods, including ticks and fleas. Rickettsiae are capable of circulating in their natural vectors through transstadial (horizontal) and transovarial (vertical) transmission and undergo dramatic niche-specific transcriptomic adaptations during their life-cycle to adjust to diverse environments in arthropod vectors and mammalian hosts. In this context, a major vacuity in our existing knowledge is the lack of understanding of regulatory mechanisms supporting their adaptive and/or pathogenic abilities in different host niches. Although small non-coding RNAs (sRNAs) are now firmly established as universal features of pathogenic bacteria and major regulators of their transcriptomes, the discovery and functional characterization of rickettsial sRNAs remained ignored until recently. We have now established and published that sRNAs in Rickettsia species are not only plentiful and functional (based on the identification of RC0877 and cydA as the respective target genes for novel R. conorii sRNAs Rc_sR35 and Rc_sR42, but are also differentially expressed in human versus tick host cells and in target organs in a mouse model of endothelial-target rickettsiosis akin to human disease. These findings serve as the foundation for a novel paradigm and genesis of our hypothesis that pathogenic rickettsiae exploit a network of sRNAs to adapt to host-specific environments (vectors vis--vis mammals) via modulation of gene expression. Having established the presence of sRNAs in both major groups of pathogenic rickettsiae, we now propose to conduct a comprehensive `compare and contrast' analysis of sRNA repertoire of R. conorii and R. typhi and their target genes during infection of human and mouse microvascular endothelium and to perform mechanistic and functional characterization of critically important rickettsial sRNAs [Aim 1]. We will next investigate their roles in rickettsial maintenance in natural vectors and vector-to-host transmission [Aim 2] and as potential determinants of virulence in the target organs of established murine models of R. conorii and R. typhi infection closely recapitulating the pathophysiology of human rickettsioses [Aim 3] . At conclusion, the proposed scientific enquiry will have a sustained positive impact through cataloging of novel rickettsial sRNAs and determination of mechanistic and functional insights into transcriptome regulation during distinct life-cycle phases of maintenance, pathogenesis, and transmission. The knowledge thus acquired will provide a platform for strategic development of new and improved treatment modalities against human rickettsial diseases.